1. Field of the Invention
The present invention relates to novel synthetic peptides. More particularly, the invention relates to novel peptides which are useful as inhibitors of mammalian collagenase.
2. Background of the Prior Art
Collagenases are proteolytic enzymes which initiate the degradation of collagen in vertebrates. In addition to their normal function in the metabolism of connective tissue and wound healing, these endoproteinases have been implicated in a number of pathological conditions such as joint destruction in rheumatoid arthritis, periodontal disease, corneal ulceration and tumor metastasis.
Of particular significance is the pathological condition caused by corneal ulceration. Corneal ulceration is caused by different agents. One such cause is alkali burning of the cornea. Although methods of treatment are known, treatment of this condition continues to be a major challenge in ophthalmology.
Many therapeutic techniques have been used in an attempt to prevent the sequellae from threatening the intergrity of the eye following a chemical injury. These include corticosteroids, heparin, collagenase inhibitors, contact lenses, fibronectin, conjunctival flaps, and corneal transplantation. Recent studies have advocated the use of sodium citrate and sodium ascorbate. Following an ocular alkali burn, a number of degradative processes occur which may result in a corneal ulcer. Several proteases, including collagenases, are released in the chemically injured cornea and account for the ulcerative process. Although the multitude of treatment modalities used in these injuries undoubtedly work by different mechanisms of action, successful management of ocular alkali burns requires the use of agents which reduce the impact of collagenase and other proteases upon the cornea.
Heretofore, the efficacy of inhibitors of collagenases for use in human corneal alkali burns is open to question. Compounds which have been tested experimentally in animals include acetylcysteine, cysteine, sodium and calcium EDTA, and penicillamine. Of these, acetylcysteine which is approved for use as a mucolytic agent, is the only collagenase inhibitor used clinically in the treatment of human alkali burns. Its efficacy has yet to be proven in a randomized clinical trial. Collagenase inhibition by the tetracycline family of antibiotics has been demonstrated in vitro and systemic tetracycline has recently been shown to inhibit alkali-induced corneal ulceration in rabbits. Thus, an adequate inhibitor of collagenase for the treatment of alkali-induced corneal ulceration has not yet been developed and is a desired goal in ophthalmology.
Another cause of corneal ulceration is infectious keratitis. Infectious keratitis is the most common and most serious of the ocular infections. The organism Pseudomonas aeruginosa (PA) is one of the leading causes of infectious keratitis. The mainstay of therapy for infectious keratitis has been antimicrobial agents, but often, even when adequate levels of antibiotics are delivered, keratitis can progress to corneal ulceration and perforation. Many organisms, such as PA, release destructive enzymes which contribute to the breakdown of the cornea. In addition to enzymes released by the organism, host-derived enzymes, such as corneal collagenase, are also involved in the pathogenesis of infectious keratitis. Again, a new treatment for this condition is clearly a major current need in opthalmology.
Another area where collagenase inhibitors may be clinically important is the control of tumor metastasis. Malignant tumor cells differ from other cancer cells in their ability to spread through the mammalian body. To do this these cells must destroy connective tissue by giving off proteolylic enzymes including collagenases. It is thus postulated that collagenase inhibitors may slow down or even stop metastasis by inhibiting these enzymes.
Collagenase inhibitors have clinical significance in the control of certain forms of dermatitis. It has been found that proteolylic enzymes, including collagenases, are involved in the destruction of skin tissue. Therefore, the administration of a collagenase inhibitor would retard and/or prevent these dermatological diseases by inhibiting these enzymes.
The mechanism of action of mammalian collagenases on the molecular level is fairly well understood. Tissue collagenases hydrolyze a specific peptide bond at a single cleavage site on each of the three collagen chains of triple helical collagen. This cleavage site is contained within the amino acid sequence Pro-Gln-Gly-Leu-(Ile)-Ala-Gly-Gln-Arg, with cleavage occurring between glycine 775 and leucine or isoleucine 776, in Types I, II and III collagen, the predominant collagen in skin, bone, tendon, dentin, fasica and cartilage. Type IV collagenase (gelatinase) degrades basement membrane (Type IV) collagen, which may be important in tumor metastasis. The collagenases are metallopeptidases which contain an essential zinc at the active site. The zinc is assumed to function by interactions with the scissile carbonyl of the substrate, thus facilitating hydrolysis of the peptide bond.
Compounds which coordinate to the zinc active site have the ability to inhibit the activity of the collagenase. Because of the clinical importance and the desirability of being able to control these enzymes' activity, there has been a widespread effort to design compounds which are capable of interacting with the enzyme binding site and preventing the enzymes' action. Consequently, there exists a number of synthetic peptides and chemically similar compounds which are claimed to have at least some effect in inhibiting the activity of mammalian collagenases. Many of these synthetic peptides are constructed so as to mimic the natural amino acid sequence flanking the collagenase cleavage site. For example, U.S. Pat. No. 4,511,504 describes a number of carboxyalkyl peptide derivatives said to have inhibitory activity. U.S. Pat. No. 4,263,293 relates to heterocyclic-containing amide compounds, U.S. Pat. No. 4,235,885 discloses mercaptoacyl amino acid derivatives, U.S. Pat. No. 4,327,111 teaches N-substituted mercaptoacyl propionamides, U.S. Pat. No. 4,382,081 describes a wide variety of mercapto amino acid derivatives, all of which appear to have some level of collagenase inhibitory activity. Similarly, U.S. Pat. No. 4,374,765 refers to the use of acyl derivatives of the peptide Gly-L-Cys-Gly-L-Gln-L-Glu-NH2. U.S. Pat. No. 4,367,233 refers to thioglycolic acid derivatives, and U.S. Pat. No. 4,361,574 teaches alkanoic acid derivatives which are useful collagenase inhibitors. U.S. Pat. No. 4,595,700 sets forth thiol-based inhibitors. European Patent Application No. 85870005.7 discloses thiopeptolide derivatives as inhibiting collagenase substrates.
Hydroxamic acid based collagenase inhibitors have also been reported in European Patent Application Nos. 87102771.0 and 86112386.7.
In U.S. Pat. Nos. 4,599,361 and 4,743,587, Dickens, et al. disclose hydroxamic acid based compounds of the formula: ##STR2## wherein R.sub.1 is C.sub.1 -C.sub.6 alkyl:
R.sub.2 is C.sub.1 -C.sub.6 alkyl, benzyl, benzyloxybenzyl, (C.sub.1 -C.sub.6 alkoxy)benzyl, benzyloxy(C.sub.1 -C.sub.6 alkyl) or hydroxybenzyl; PA1 A is either (CHR.sub.3 -CHR.sub.4), or (CR.sub.3 -CR.sub.4); where R.sub.3 is hydrogen, C.sub.1 -C.sub.6 alkyl, phenyl or phenyl (C.sub.1 -C.sub.6 alkyl), R.sub.4 is hydrogen, C.sub.1 -C.sub.6 alkyl, phenyl (C.sub.1 -C.sub.6 alkyl), cycloalkyl or cycloalkyl (C.sub.1 -C.sub.6 alkyl). PA1 R.sub.1 is C.sub.2 -C.sub.5 alkyl; PA1 R.sub.2 is a natural amino acid having a functional group containing amino or carboxy with the proviso that R.sub.2 is not hydrogen or methyl; PA1 R.sub.3 is hydrogen, amino, hydroxy, mercapto, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy, C.sub.1 -C.sub.6 alkylthio, aryl (C.sub.1 -C.sub.6 alkyl), amino (C.sub.1 -C.sub.6 alkyl), hydroxy (C.sub.1 -C.sub.6 alkyl), mercapto (C.sub.1 -C.sub.6 alkyl) or carboxy (C.sub.1 -C.sub.6 alkyl), wherein the amino, hydroxy, mercapto and carboxyl groups may be protected by an acylated amino group; PA1 R.sub.4 is hydrogen or methyl; PA1 R.sub.5 is hydrogen, C.sub.1 -C.sub.6 methyl, C.sub.1 -C.sub.6 alkoxy, C.sub.1 -C.sub.6 alkyl, di(C.sub.1 -C.sub.6 alkoxy)carbonyl, arylmethoxycarbonyl, (C.sub.1 -C.sub.6 alkyl)aminocarbonyl or arylaminocarbonyl; PA1 R.sub.6 is hydrogen or methyl; PA1 R.sub.2 and R.sub.4 may be taken together to form a (CH.sub.2).sub.n ring wherein n is 4 to 11; or R.sub.4 and R.sub.5 may be taken together to form a (CH.sub.2).sub.3 ring. PA1 R.sub.2 and R.sub.3 are the same or are different and represent hydrogen or a C.sub.1 -C.sub.10 saturated alkyl; and ##STR5## PA1 Z represents an amino radical or an alkylaminol in which the alkyl group contains 1 or 2 atoms of carbon and is substituted by a phenyl or a trifluorophenyl. PA1 R.sub.2 and R.sub.3 may be the same or different and may be an amino acid residue chosen from the following: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, glutamic acid and aspartic acid, such that each amino residue, however, must not have an acidic terminus, but optionally may have an acidic side chain; PA1 n is either 1 or 2; and PA1 A is hydrogen or is --CHR.sub.4 --CO--NH.sub.2, where R.sub.4 is an amino acid residue. PA1 R.sub.2 is aryl lower alkyl or heterocyclic lower alkyl; said R.sub.2 being unsubstituted or mono- or di-substituted with chloro, fluoro, bromo, nitro, carboxy, lower carbalkoxy, cyano, lower alkanoyl, trifluoromethyl, lower alkyl, hydroxy, lower alkoxy, formyl, amino, lower alkylamino, dilower alkylamino, mercapto, lower alkylthio or mercapto lower alkyl; ##STR9## AA.sub.1 is an amino acid residue; X is a chemical bond, lower alkylene, ##STR10## R.sub.9 and R.sub.10 are independently hydrogen, methyl or ethyl D, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently hydrogen or lower alkyl; and PA1 m is 1, 2, or 3, with the proviso that when B is ##STR11## and X is a chemical bond or lower alkylene then R.sub.2 is not unsubstituted benzyl or benzyl monosubstituted with hydroxy, or lower alkoxy and with the further proviso that when B is ##STR12## and X is ##STR13## then R.sub.2 is not unsubstituted indole or unsubstituted benzyl or benzyl monosubstituted with hydroxy or lower alkoxy. PA1 Ala--Alanine PA1 Thr--Threonine PA1 Gly--Glycine PA1 Cys--Cysteine PA1 His--Histidine PA1 Met--Methionine PA1 Leu--Leucine PA1 Pro--Proline PA1 Ile--Isoleucine PA1 Ser--Serine PA1 Asp--Aspartic Acid PA1 Glu--Glutamic Acid PA1 Phe--Phenylalanine PA1 Trp--Tryptophan PA1 Lys--Lysine PA1 Arg--Arginine PA1 Asn--Asparagine PA1 Gln--Glutamine PA1 Tyr--Tyrosine PA1 Val--Valine
These hydroxamic acid based compounds are alleged to be collagenase inhibitors.
Handa, et al. in European Patent Application No. 0 236 872 disclose compounds having the formula: ##STR3## wherein A is either HO--NH--CO or HCO--N(OH)--;
The above compounds disclosed by Handa, et al. are alleged to inhibit collagenase.
In European Patent Application No. 0 262 053, Fournie-Zaluski, et al. disclose compounds of the formula: ##STR4## wherein R.sub.1 is a saturated C.sub.1 -C.sub.10 alkyl;
It is alleged that compounds of this formula inhibit collagenase.
Cartwright, et al. in European Patent Application No. 274 453 discloses compounds of the formula: ##STR6## wherein W represents valine, lysine, norleucine or methionine; and
It is alleged that these compounds inhibit collagenase.
In U.S. Pat. Nos. 4,687,841 and 4,720,486, Spilburg, et al. disclose tripeptides of the formula: EQU HO-NH-Gly-Leu-Pro-R
wherein R represent hydrogen, agaroses or an .alpha.-amino protecting group such as an alkanoyl, aroyl or cycloalkanoyl.
These compounds allegedly function either as collagen inhibitors or as affinity resins for the purification of vertebrate collagenase.
Wolanin, et al. in U.S. Pat. No. 4,771,038 disclose compounds having the formula: ##STR7## wherein R.sub.1 is C.sub.2 -C.sub.7 alkyl;
Compounds of this formula are alleged to inhibit metalloproteases, particularly endopeptidases such as collagenase.
In addition to patents, the scientific literature also contains references to many collagenase inhibiting compounds. Clark, et al. (Life Sciences 37: 575-578 (1985)) refer to N[[5-chloro-2-benzothiazolyl)thiophenyl]acetyl]-L-cysteine, said to be a powerful mammalian collagenase inhibitor. Deleaisse, et al. (Biochem Biophys. Res. Comm. 133: 483-490, 1985) also refer to an inhibitor N-[3-N-(benzyloxy-carbonyl)-amino-1-(R)-carboxypropyl]-L-leucy-1-O-methyl- L-tyrosine-N-methylamide. Gray, et al. (Biochem. Biophys. Res. Comm. 101: 1251-1258, 1981) disclose a number of thiol-containing analogues of the collagen cleavage site. Additional thiol-containing peptides are disclosed by Gray, et al. in J. Cell Biochem., 32: 71-77, 1986. Carboxyalkyl peptide analogues are described in Gray, et al. in Federation Proc. 44: 1431, 1985. Miller, et al. and Gray, et al. also disclose thiol-containing peptides in abstracts. [Fed. Proc. 45: 1859 (1986) and FASEB J. 2: A345 (1988), respectively]. Mookhtiar, et al. also discloses phosphonamidate inhibitors of collagenase. (see Biochemistry, 26, 1962 (1987)).
Despite the large number of compounds showing inhibitory properties, the therapeutically useful commercially available compounds are very few in number and are not altogether satisfactory in all respects for clinical use. Therefore, a continued need exists for an extremely potent and highly specific collagenase inhibitor which will have widespread therapeutic and commercial application. It has now been discovered that a small class of novel hydroxamic acid-containing tripeptides provides a level of collagenase inhibition not heretofore observed in the known inhibitory compounds.